Twenty-five years since the discovery of HIV-1, the underlying biological mechanism(s) of male-to-female sexual transmission remain unclear. To initiate infection, HIV must traverse the protective outer genital epithelial barriers, establishing infection in underlying immunocompetent target cells. To study the nteractions of individual HIV virions with mucosal epithelial barriers, we developed a novel system comprising genital tissue explants and fluorescent microscopy. A difficult aspect of fluorescent microscopic analysis of tissue sections is the high background autofluorescence. To avoid this problem, we utilize HIV abeled with a photoactivatable form of green fluorescent protein (GFP). Tissue sections are initially scanned to define background autofluorecence. The field of view is then photoactivated, followed by a second scan. Viral particles appear as new signals observed only after photoactivation. Using this system we find that HIV can enter both the squamous ectocervical epithelium and the columnar endocervical epithelium in human explants. Future studies will analyze vaginal and uterine tissue. Virions are observed penetrating to depths that would facilitate contact with HIV targets such as Langerhans cells, macrophages, dendritic cells and CD4+ T cells. We find that the virus is primarily localized in interstitial spaces between cells in the squamous epithelium. This suggests that virus has the ability to move within the intact mucosal epithelia to encounter potential target cells of infection. Related studies will determine how cell-associated virus interacts with the tissues of the female genital tract. The specific aims seek to 1) define the interaction of HIV with the intact squamous epithelium of the ectocervix and vagina, 2) define the interaction of HIV with the intact columnar epithelium of the endocervix and uterus, 3) characterize the interaction of HIV with cervical mucous, and 4) determine how exogenous factors such as inflammation and other local environmental factors modulate the interaction of HIV with the mucosal epithelium leading to increased or decreased infection. The completion of these aims will increase our understanding of the underlying mechanism(s) of HIV sexual transmission. This information can then be utilized in the development of vaccines and microbicides designed to prevent HIV. RELEVANCE (See instructions): There is currently a large effort to develop microbicides and vaccines to prevent the sexual transmission of HIV. Yet we currently do not understand in much detail how HIV is sexually transmitted. The studies propose here seek to reveal novel details of how HIV interacts with the human female genital tract leading to infection. This information will be invaluable in the development of strategies to prevent HIV transmission.